Canine platelets express functional Toll-like receptor-4: lipopolysaccharide-triggered platelet activation is dependent on adenosine diphosphate and thromboxane A2 in dogs.

BACKGROUND: Functional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A2 (TxA2). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS). RESULTS: We discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B2 secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA2 pathway. CONCLUSION: Expression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA2 pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.

A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy.

BACKGROUND: Canine neutrophils release neutrophil extracellular traps (NETs) in response to lipopolysaccharide but NETs from clinical septic dogs had not been identified. The primary aim is to describe the methodology of identifying and quantifying neutrophil extracellular traps (NETs) in cytology samples of septic foci in dogs with sepsis using immunofluorescence microscopy. Cytology samples including endotracheal tracheal wash (ETW), bronchoalveolar lavage (BAL), abdominal and pleural effusion collected from 5 dogs (3 septic, 2 non-septic) were fixed, permeabilized and stained for myeloperoxidase (MPO), citrullinated histone H3 (citH3) and cell-free DNA (cfDNA). Fluorescence microscopy was used to identify and quantify NETs in 10 random views at 40× magnification. NETs were identified based on co-localization of MPO, citH3 and cfDNA. NETs were quantified as a ratio (number of NETs: number of neutrophils). Neutrophils were identified based on cytoplasmic MPO, cellular diameter and nuclear morphology. RESULTS: NETs were identified and quantified in all cytology samples collected from septic dogs. A small number of NETs was documented in one dog with sterile chronic bronchitis. No NETs were found in sterile abdominal effusion collected from one dog with congestive heart failure. CONCLUSIONS: Immunofluorescence microscopy could be a useful tool for the study of NETs in dogs with clinical sepsis.

Early Life Wildfire Smoke Exposure Is Associated with Immune Dysregulation and Lung Function Decrements in Adolescence.

The long-term health effects of wildfire smoke exposure in pediatric populations are not known. The objectives of this study were to determine if early life exposure to wildfire smoke can affect parameters of immunity and airway physiology that are detectable with maturity. We studied a mixed-sex cohort of rhesus macaque monkeys that were exposed as infants to ambient wood smoke from a series of Northern California wildfires in the summer of 2008. Peripheral blood mononuclear cells (PBMCs) and pulmonary function measures were obtained when animals were approximately 3 years of age. PBMCs were cultured with either LPS or flagellin, followed by measurement of secreted IL-8 and IL-6 protein. PBMCs from a subset of female animals were also evaluated by Toll-like receptor (TLR) pathway mRNA analysis. Induction of IL-8 protein synthesis with either LPS or flagellin was significantly reduced in PBMC cultures from wildfire smoke-exposed female monkeys. In contrast, LPS- or flagellin-induced IL-6 protein synthesis was significantly reduced in PBMC cultures from wildfire smoke-exposed male monkeys. Baseline and TLR ligand-induced expression of the transcription factor, RelB, was globally modulated in PBMCs from wildfire smoke-exposed monkeys, with additional TLR pathway genes affected in a ligand-dependent manner. Wildfire smoke-exposed monkeys displayed significantly reduced inspiratory capacity, residual volume, vital capacity, functional residual capacity, and total lung capacity per unit of body weight relative to control animals. Our findings suggest that ambient wildfire smoke exposure during infancy results in sex-dependent attenuation of systemic TLR responses and reduced lung volume in adolescence.

Intra-articular use of a platelet-rich product in normal horses: clinical signs and cytologic responses.

OBJECTIVES: (1) To report the clinical and synovial effects of a platelet-rich product (PRPr) in normal equine joints, (2) to assess the persistence of platelets within synovial fluid after intra-articular injection, (3) to compare responses to different preparations of that product, and (4) to evaluate a gravity filtration system for PRPr preparation in horses. STUDY DESIGN: Experimental. METHODS: A platelet-rich saline product (PRPr) was prepared from 7 normal horses using a proprietary preparation device and was divided into 3 treatments: resting, CaCl2 -activated (23 mM, final), and bovine thrombin-activated (10 U/mL, final). Each horse had 3 concurrent randomly assigned intra-articular PRPr treatments administered in their metacarpophalangeal/metatarsophalangeal joints; the fourth limb was injected with saline (0.9% NaCl) solution as a control. Clinical assessments, cytologic analysis of synovial fluid and hemograms were performed at 6, 24, 48, and 96 hours after injection. PRPr composition and growth factor content were analyzed. RESULTS: The gravity filtration system produced a moderately concentrated PRPr. At 6 and 24 hours, when compared to control values, all PRPr treatments caused a significant increase in synovial WBC concentration (P < .0059) and neutrophil percentage (P < .0005). Bovine thrombin-activated PRPr injection consistently caused increased effusion scores and periarticular signs. At all time points, the synovial WBC concentration after thrombin-activated PRPr was significantly greater (P < .001) than for the control, CaCl2 -activated or resting PRPr. Intact platelets could be observed in synovial fluid for up to 5 days after intra-articular PRPr injection. CONCLUSIONS: Resting and CaCl2 -activated PRPr may be safely used to treat equine joints, but bovine thrombin activation is not recommended at 10 U/mL. A PRPr can be prepared using a gravity filtration system, eliminating the need for centrifugation.

Seasonal influences on CAPs exposures: differential responses in platelet activation, serum cytokines and xenobiotic gene expression.

Increasing evidence suggests a role for a systemic pro-coagulant state in the pathogenesis of cardiac dysfunction subsequent to inhalation of airborne particulate matter (PM). We evaluated platelet activation, systemic cytokines and pulmonary gene expression in mice exposed to concentrated ambient particulate matter (CAPs) in the summer of 2008 (S08) and winter of 2009 (W09) from the San Joaquin Valley of California, a region with severe PM pollution episodes. Additionally, we characterized the PM from both exposures including organic compounds, metals, and polycyclic aromatic hydrocarbons. Mice were exposed to an average of 39.01 µg/m(3) of CAPs in the winter and 21.7 µg/m3 CAPs in the summer, in a size range less than 2.5 µm for 6 h/day for 5 days per week for 2 weeks. Platelets were analyzed by flow cytometry for relative size, shape, CD41, P-selectin and lysosomal associated membrane protein-1 (LAMP-1) expression. Platelets from W09 CAPs-exposed animals had a greater response to thrombin stimulation than platelets from S08 CAPs-exposed animals. Serum cytokines were analyzed by bead based immunologic assays. W09 CAPs-exposed mice had elevations in IL-2, MIP-1α, and TNFα. Laser capture microdissection (LCM) of pulmonary vasculature, parenchyma and airways all showed increases in CYP1a1 gene expression. Pulmonary vasculature showed increased expression of ICAM-1 and Nox-2. Our findings demonstrate that W09 CAPs exposure generated a greater systemic pro-inflammatory and pro-coagulant response to inhalation of environmentally derived fine and ultrafine PM. Changes in platelet responsiveness to agonists, seen in both exposures, strongly suggests a role for platelet activation in the cardiovascular and respiratory effects of particulate air pollution.

Response of Northern Elephant Seal platelets to pressure and temperature changes: a comparison with human platelets.

Mammalian blood platelets are activated by physiological agonists such as collagen or thrombin, or by physical stimuli such as cold temperatures and rapid decompression. Marine mammals regularly experience cold temperatures, high pressures and rapid decompression while diving, yet do not appear to suffer from thrombotic events during routine dive activity. We evaluated the effects of cold temperature and high pressure excursions on Northern Elephant Seal (NES) platelets and compared NES platelet response to that of human platelets subjected to identical stimuli. NES platelets undergo cold-induced activation when chilled to 4 °C, and 3 distinct phase transitions can be measured using Fourier Transform Infrared Spectroscopy. NES platelet membrane lipid composition was determined using thin layer chromatography and NES platelets were found to have three times the amount of cholesterol (21% by weight) as human platelets. When exposed to high pressure-rapid decompression excursion, NES platelets did not undergo morphological shape change nor bind increased amounts of fibrinogen, while human platelets were significantly activated by the same excursion. These results demonstrate that while NES platelets are activated by the physical stimulus of cold temperatures, they are resistant to decompression-induced activation. We suggest that the composition of NES platelet membranes may play an important role in preventing pressure-related activation.

Activation of equine platelet-rich plasma: comparison of methods and characterization of equine autologous thrombin.

OBJECTIVE: To investigate and compare clinically relevant Platelet-rich plasma (PRP) activation methods. STUDY DESIGN: Experimental. METHODS: PRP was prepared from 6 equine subjects. Activation of the PRP was performed by 4 methods (autologous thrombin, bovine thrombin, calcium chloride (CaCl(2) ), or freeze-thaw). The resultant PDGF-BB (where PDGF is platelet-derived growth factor) and TGFß1 (where TGFß is transforming growth factor beta) levels in PRP releasates were quantified by Enzyme-linked immunosorbent assay (ELISA) and compared. Growth factor contents were also compared between platelet-rich clots produced by thrombin or CaCl(2) . The composition and function of equine autologous thrombin were characterized by Western blot analysis and platelet aggregometry. RESULTS: CaCl(2) (23 mM) activation of PRP yielded significantly greater PDGF release than did any other method. TGFß release was comparable after PRP activation by CaCl(2) , bovine thrombin, and freeze thaw. Autologous thrombin was significantly less effective than all other activation methods in eliciting platelet growth factor release and induced significantly less platelet aggregation than bovine thrombin at 5 U/mL. Clots retained substantial concentrations of growth factor, and the amount in the releasate versus the clot differed between activation methods. CONCLUSIONS: PRP activation methods differ in terms of growth factor output as well as logistical considerations. Autologous thrombin is not recommended for PRP activation. CaCl(2) (23 mM) is an effective and inexpensive method of PRP activation. The PRP releasate derived from CaCl(2) activation contains 80% of the total PDGF content and is easily produced, making it a convenient product for clinical use.

Effects of preparation method, shear force, and exposure to collagen on release of growth factors from equine platelet-rich plasma.

OBJECTIVE: To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP). SAMPLE POPULATION: PRP obtained from 6 horses. PROCEDURES: PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 µg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor ß, isoform 1 (TGFß1); and insulin-like growth factor, isoform 1 (IGF-1) were quantified by use of ELISAs. Statistical analysis was conducted via repeated-measures ANOVA. RESULTS: Platelet numbers were significantly higher in tube-prepared PRP than in automated-prepared PRP Growth factor concentrations did not differ significantly between preparation methods. Mean PDGF-BB concentration ranged from 134 to 7,157 pg/mL, mean TGFß1 concentration ranged from 1,153 to 22,677 pg/mL, and mean IGF-1 concentration ranged from 150 to 280 ng/mL. Shear force did not affect growth factor concentrations. Dose-dependent increases in PDGF-BB and TGFß1 were detected in response to collagen, but equalled only 10% of the estimated total platelet content. Concentrations of IGF-1 were not significantly different among treatments and negative or positive control treatments. Serum concentrations of PDGF-BB and TGFß1 exceeded concentrations in PRP for most treatment conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Release of growth factors from equine PRP was negligible as a result of the injection process alone. Investigation of platelet-activation protocols is warranted to potentially enhance PRP treatment efficacy in horses.

Genetics of equine bleeding disorders.

Genetic bleeding disorders can have a profound impact on a horse's health and athletic career. As such, it is important to understand the mechanisms of these diseases and how they are diagnosed. These diseases include haemophilia A, von Willebrand disease, prekallikrein deficiency, Glanzmann's Thrombasthenia and Atypical Equine Thrombasthenia. Exercise-induced pulmonary haemorrhage also has a proposed genetic component. Genetic mutations have been identified for haemophilia A and Glanzmann's Thrombasthenia in the horse. Mutations are known for von Willebrand disease and prekallikrein deficiency in other species. In the absence of genetic tests, bleeding disorders are typically diagnosed by measuring platelet function, von Willebrand factor, and other coagulation protein levels and activities. For autosomal recessive diseases, genetic testing can prevent the breeding of two carriers.

Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content.

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 degrees C, but not at 37 degrees C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets ( approximately 15 mol%), appears to be crucial for the formation of large domains during cooling.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds.

OBJECTIVE: To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS: 13 healthy adult Thoroughbreds. PROCEDURES: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS: Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

Comparison of nonparametric and parametric methods for time-frequency heart rate variability analysis in a rodent model of cardiovascular disease.

The aim of time-varying heart rate variability spectral analysis is to detect and quantify changes in the heart rate variability spectrum components during nonstationary events. Of the methods available, the nonparametric short-time Fourier Transform and parametric time-varying autoregressive modeling are the most commonly employed. The current study (1) compares short-time Fourier Transform and autoregressive modeling methods influence on heart rate variability spectral characteristics over time and during an experimental ozone exposure in mature adult spontaneously hypertensive rats, (2) evaluates the agreement between short-time Fourier Transform and autoregressive modeling method results, and (3) describes the advantages and disadvantages of each method. Although similar trends were detected during ozone exposure, statistical comparisons identified significant differences between short-time Fourier Transform and autoregressive modeling analysis results. Significant differences were observed between methods for LF power (p ≤ 0.014); HF power (p ≤ 0.011); total power (p ≤ 0.027); and normalized HF power (p = 0.05). Furthermore, inconsistencies between exposure-related observations accentuated the lack of agreement between short-time Fourier Transform and autoregressive modeling overall. Thus, the short-time Fourier Transform and autoregressive modeling methods for time-varying heart rate variability analysis could not be considered interchangeable for evaluations with or without interventions that are known to affect cardio-autonomic activity.

Equine platelet concentrate preparation and validation.

BACKGROUND: Development of equine platelet concentrate (PC) would aid management of cases requiring transfused platelets (PLTs), where adminstration of whole-blood or platelet-rich plasma (PRP) might be contraindicated. OBJECTIVES: To test and validate a method for production of an equine PRP-PC product. ANIMALS: Six healthy Thoroughbred geldings from a research herd. METHODS: In this prospective experimental study, whole blood was collected and processed through multiple centrifugation steps to yield 120 mL of PC. The PC was stored at 22°C and gently and continuously agitated. Measurements of PLT count, pH, and concentrations of glucose, lactate, electrolytes, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST), as well as partial pressures of oxygen and carbon dioxide were performed on days 0, 1, 2, 3, 5, and 7. Platelet aggregometry and bacterial culture were also performed. RESULTS: The PC always had a PLT count of ≥550 × 103 cells/µL. Aggregometry graph amplitude (P < .0001) and area under the curve (P < .05) significantly decreased over time. Sodium, chloride, lactate (P < .0001), and oxygen (P < .01) concentrations significantly increased over time. pH (P < .001), glucose and bicarbonate concentrations (P < .0001) significantly decreased over time. There was no significant difference in potassium concentration, PLT count, LDH and AST activities and no bacterial growth from culture. CONCLUSIONS AND CLINICAL IMPORTANCE: The described technique yielded a PC that meets the standards of the American Association of Blood Banks for human PC.

Pharmacokinetics and competitive pharmacodynamics of ADP-induced platelet activation after oral administration of clopidogrel to horses.

OBJECTIVE: To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS: Compared with results for preadministration samples, platelet aggregation in response to 12.5µM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25µM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20µM) and prostaglandin E1 (3.3µM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505-512).

Assessment of a dimethyl sulfoxide-stabilized frozen canine platelet concentrate.

OBJECTIVE: To assess platelet count, mean platelet volume (MPV), metabolic characteristics, and platelet function in a dimethyl sulfoxide (DMSO)-stabilized canine frozen platelet concentrate (PC). SAMPLE POPULATION: 11 units of a commercial frozen PC in 6% DMSO and fresh platelet-rich plasma from 6 healthy control dogs. PROCEDURES: PCs were thawed, and the following data were collected: thaw time, platelet count, MPV, pH, PCO2, and PO2 and HCO3-, glucose, and lactate content. Phosphatidylserine translocation was determined by use of flow cytometry. Fresh platelet-rich plasma from healthy dogs served as a source of control platelets for flow cytometric analysis. RESULTS: At thaw, the platelet count in the frozen PC ranged from 243,000 to 742,000 platelets/microL. Median platelet count of paired samples was 680,000 platelets/microL and decreased significantly to 509,000 platelets/microL at 2 hours after thaw. Median MPV at thaw was 11.15 femtoliters and was stable after 2 hours. Compared with fresh platelets, frozen PC had increased amounts of phosphatidylserine in the outer leaflet of the platelet membrane in the resting (ie, not treated with thrombin) state (19% vs 99%, respectively) and alterations in cellular morphology, all of which were consistent with platelet activation. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this in vitro study indicated that there was a decrease in platelet quantity and function as well as an increase in platelet activation during the freeze-and-thaw process in DMSO-stabilized canine frozen PC. In vivo effects on PC remain to be determined.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

Platelet function in clinically healthy cats and cats with hypertrophic cardiomyopathy: analysis using the Platelet Function Analyzer-100.

BACKGROUND: There is currently no simple analytical tool for the evaluation of hypercoagulability in cats. The Platelet Function Analyzer-100 (PFA-100; Dade Behring Inc., Deerfield, IL, USA) is a bench-top machine that evaluates platelet function by measuring closure time (CT) in citrated whole blood under high shear conditions. We hypothesized that cats with hypertrophic cardiomyopathy (HCM) have up-regulated platelet function, which shortens their CT and increases their risk for thromboembolic events. OBJECTIVES: The goals of this study were to: (1) establish a feline reference interval for CT using the PFA-100, (2) measure CT in blood from cats with HCM, and (3) determine if there is a measurable difference between the CT of healthy cats compared with cats with HCM. METHODS: Citrated blood samples from 42 clinically healthy cats and 30 cats with HCM were analyzed according to manufacturer's specifications. CT was measured in triplicate and the mean value was used for analysis. Transformed data were compared between clinically healthy cats and cats with HCM using a Student's t-test, and among cats with mild, moderate, or severe HCM using ANOVA. RESULTS: The median CT of clinically healthy cats was 64 seconds (range 43-176 seconds). The median CT of cats with HCM was 74 seconds (range 48-197 seconds). There was no significant difference in CT between cats with HCM and clinically healthy cats. There also were no significant differences in cats with mild, moderate, or severe HCM. CONCLUSIONS: A feline reference interval for PFA-100 CT will be useful in future studies of platelet function in cats. Cats with HCM do not have shorter CTs when compared with clinically healthy cats.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.